Methods for cyclic nucleotide determination

ABSTRACT

The present invention relates in general to cellular analysis tools and more particularly to methods for detecting or determining cyclic nucleotide concentrations in samples. Samples containing cyclic nucleotides may be contacted with a cyclic nucleotide-dependent protein kinase and a detection system which includes a substrate for the cyclic nucleotide-dependent protein kinase. The activities in cyclic nucleotide related pathways may be measured using the detection system.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/710,087, filed Feb. 22, 2010, which is a continuation of U.S.application Ser. No. 11/634,756, filed Dec. 6, 2006 now abandoned, whichclaims the benefit of priority to U.S. Provisional Application No.60/742,922, filed Dec. 6, 2005, the entire contents of all of which arefully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates in general to cellular analysis tools andmore particularly to methods for detecting or determining cyclicnucleotide concentrations in samples.

BACKGROUND OF THE INVENTION

The second messengers, adenosine 3′, 5′cyclic monophosphate (cAMP) andguanosine 3′, 5′cyclic monophosphate (cGMP), are important intracellularmediators of a variety of cellular functions including cell growth,differentiation, apoptosis, and cell death. Production of cAMP iscontrolled through the adenylyl cyclase family of enzymes, which convertadenosine triphosphate (ATP) to cAMP and inorganic pyrophosphate (PPi).The adenylyl cyclases are activated or inhibited via direct interactionwith membrane bound G-protein coupled receptor (GPCR) α-subunits. Whenan α-subunit of a stimulatory GPCR is activated, designated G_(αs),adenylyl cyclase converts ATP to cAMP and PPi. Conversely, when anα-subunit of an inhibitory GPCR is activated, designated G_(αi), aninhibitory effect on adenylyl cylase is exerted and the conversion ofATP to cAMP and PPi is not realized. G-protein coupled receptors play aprominent role in a wide variety of biological processes such asneurotransmission, cardiac output, and pain modulation. Their importancein developing new medically useful compounds is well understood; as suchthey are highly targeted in drug discovery research.

The intracellular concentration of cAMP is also affected by anothergroup of enzymes, cyclic nucleotide phosphodiesterases (PDE), whichcatalyze the hydrolysis of cAMP to AMP and cyclic cGMP to GMP.Phosphodiesterases function in conjunction with adenylyl cyclases andguanylate cyclases to regulate the amplitude and duration of cellsignaling mechanisms that are mediated by cAMP and cGMP.Phosphodiesterases therefore regulate a wide range of importantbiological responses to first messengers such as hormones, light, andneurotransmitters. There are two classes of PDEs; Class I are found inthe cytoplasm or bound to intracellular organelles or membranes of alleukaryotic cells, whereas Class II PDEs are not well characterized andhave only been found in lower eukayotes. Cellular responses controlledby Class I phosphodiesterases, through control of cAMP and cGMPconversion, include neuronal responses, aldosterone production,regulation of platelet aggregation, insulin regulation, emesis,regulation of smooth muscle tension, visual phototransduction, andmodulation of T-cell responsiveness. Numerous clinically importantcompounds are known to inhibit phosphodiesterases including; rolipram,theophylline, and sildenafil. Therefore, inhibitors ofphosphosdiesterases are also important targets in drug discovery.

The second messenger cAMP is known to activate cAMP dependent proteinkinase (PKA). Mammalian holo-PKA is a tetramer, made up of tworegulatory and two catalytic subunits. cAMP binds to the regulatorysubunits, thereby dissociating holo-PKA into its catalytic andregulatory subunits. Once released, the free catalytic subunits arecapable of phosphorylating a multitude of cellular proteins, therebycausing changes in cellular functions such as muscle contraction,activation of cell cycle, activation of transcriptional activity, andDNA processing.

Because the activation or inhibition of GPCR and subsequent activationor inhibition of adenylyl cyclase results in an increase or decrease inintracellular cAMP, agents that affect their activity are importanttargets for drug discovery. Drugs that target GPCR account for many ofthe medicines sold worldwide due to the tremendous variety of biologicalprocesses relating to G-protein coupled receptors. Examples of drugsthat influence GPCR include Claritin® and Alavert® (loratadine) whichare used for relieving allergy symptoms, Paxil® (paroxetine HCl) forrelief of depression, and Vasotec® (enalapril maleate) for relief ofhypertension. Because of their importance, various GPCR assays have beendeveloped to determine the effect of agonists and antagonists on thesesystem components, mainly by assaying for the increase or decrease incAMP levels. Limitations of these methods include non-homogeneous assaysthat require multiple dispensing steps, long incubation times, and theneed for expensive equipment.

Therefore, what are needed are assays that require less manipulationthan currently available technologies (e.g. two steps or less), assaysthat provide shorter incubation times (e.g., less than 1 hour), andassays that utilize low cost equipment while maintaining high throughputsystem (HTS) capabilities (e.g., luminescent based equipment). Suchstreamlining and cost effectiveness will allow for faster and easierevaluation of targets for drug discovery. Furthermore, luminescent basedassays are not prone to interference from fluorescence; that is usefulin screening large libraries of chemicals to discover the next potentialdrug.

SUMMARY OF THE INVENTION

The present invention relates in general to cellular analysis tools andmore particularly to methods for detecting or determining cyclicnucleotide concentrations in samples.

Cyclic nucleotides, such as cAMP and cGMP, increase or decrease inresponse to a variety of substances that interact with cellularproteins. The methods described herein provide for the detection of suchchanges. In one embodiment, the methods described herein permit cyclicnucleotides to be detected and correlated with the effect of a stimuluson cellular proteins.

In one embodiment, methods as described herein monitor the binding ofcyclic nucleotides to an enzyme that is dependent upon cyclic nucleotidebinding in order to activate the enzyme (e.g. cAMP dependent proteinkinase, or PKA). For example, once cAMP binds to PKA, PKA transfers aphosphate from adenoside triphosphate (ATP) to a suitable PKA substrate(e.g. Kemptide). The phosphorylation event is detected by various knownmethods, and the output of each detection method is correlated to theamount of cyclic nucleotide present in a sample. Suitable detectionmethods include, but are not limited to, methods based on luminescence,radioactivity, and fluorescence.

In one embodiment, a method to determine adenylyl cyclase activity in asample is provided. Said method utilizes the activation of PKA toprovide an activity that can be detected, measured and subsequentlycorrelated to adenylyl cyclase activity. For example, if adenylylcyclase is stimulated, cAMP is produced which activates PKA, whoseactivity is detected and correlated to adenylyl cyclase activity.

In another embodiment, a method to determine phosphodiesterase activityin a sample is provided. Said method utilizes the activation of PKA toprovide an activity that is detected, measured, and subsequentlycorrelated to phosphodiesterase activity. For example, if aphosphodiesterase is inhibited, cAMP is not converted to AMP or cGMP isnot converted to cGMP, therefore cAMP and cGMP can activate PKA, whoseactivity is detected and correlated to phosphodiesterase activity.

In further embodiments, methods for monitoring the activation of aG-protein coupled receptor (GPCR) by an agonist, or its inhibition by anantagonist, are provided. For example, the level of cAMP found uponaddition of agonist or antagonist to a sample comprising a GPCR isdetected and measured through the activation of PKA. Such activity (orlack thereof) is detected by a measurable output that is correlated tocAMP levels or amounts.

In one embodiment, samples used in practicing the methods as describedherein comprise a lysate. In some embodiments, the sample lysate isderived from prokaryotes or eukaryotes such as bacteria, yeast ormammalian cells. In some embodiments, said sample comprises plasmamembranes, cellular membranes, and/or organellar membranes. Membranepreparations as described herein have furnished unexpected results, suchthat the membrane preparations maintain the integrity and functionalityof processes, proteins and receptors (Examples 9-11) associated with themembranes. This allows for targeted membrane functional assays to beperformed using the methods as described herein, without accompanyingcell lysate components found in a normal cell lysate.

Measurable output may be in the form of bioluminescence,chemiluminescence, radioactivity, or differential output based ondifferent fluorescence technologies (e.g. fluorescence polarization,fluorescence resonance energy transfer, and immunoassay). In oneembodiment, the measurable output is in the form of bioluminescence. Forexample, the coleopteran (firefly) luciferase enzyme utilizes ATP andother factors to convert beetle luciferin to oxyluciferin, a byproductof the reaction being light. Once PKA is activated, the amount of PKAactivation is dependent on the amount of cAMP present, PKA utilizes aphosphate from ATP to phosphorylate a receptive substrate, therebycausing the concentration of ATP to decrease in a sample, therebycausing a decrease in luminescence, or light output. As such, as cAMPconcentration in a sample increases a reciprocal decrease inluminescence is seen which is correlated to the amount of cAMP, adenylylcyclase, and/or GPCR activity present in the initial sample.

In one embodiment, the present invention provides a method fordetermining the amount of cyclic nucleotides in a sample comprising asample with may contain a cyclic nucleotide, adding to said sample aninactive enzyme capable of being activated by said cyclic nucleotide,adding a detection system capable of detecting the activity of saidactivated enzyme and generating a detectable signal, and determining theamount of cyclic nucleotide present in said sample based on said signal.In some embodiments, said sample comprises a lysate. In someembodiments, the sample lysate is derived from bacteria, yeast ormammalian cells. In some embodiments, said sample comprises plasmamembranes, cellular membranes, and/or organellar membranes. In someembodiments, said cyclic nucleotide is cAMP or cGMP. In someembodiments, said inactive enzyme is a cAMP dependent protein kinase ora cGMP dependent protein kinase. In some embodiments, said detectionsystem comprises a substrate capable of being phosphorylated by PKA orPKG. In some embodiments, said substrate comprises SEQ ID NO: 1. In someembodiments, said detection system further comprises an enzyme capableof utilizing ATP to generate a luminescent signal wherein said enzyme isluciferase. In some embodiments, said substrate comprises aradioactively labeled biotinylated substrate further comprising SEQ IDNO: 1. In some embodiments, said detection system further comprises astreptavidin coated binding surface. In some embodiments, said substratecomprises a fluorescently labeled substrate further comprising SEQ IDNO: 1, wherein said fluorescent label is preferentially rhodamine. Insome embodiments, the method of the present invention further comprisesthe addition of one or more inhibitors of phosphodiesterases, and/or theaddition of an agonist or antagonist capable of affecting cyclicnucleotide amounts in said sample. In some embodiments, said agonist orantagonist modulates adenylyl cyclase activity and/or GPCR activityand/or PDE activity.

In one embodiment, the present invention provides a method fordetermining adenylyl cyclase activity in a sample comprising a samplethat may contain adenylyl cyclase, adding to said sample an inactiveenzyme capable of being activated by cAMP, adding a detection systemcapable of detecting the activity of said activated enzyme andgenerating a detectable signal, and determining adenylyl cyclaseactivity present in said sample based on said signal. In someembodiments, said sample comprises a lysate. In some embodiments, thesample lysate is derived from bacteria, yeast or mammalian cells. Insome embodiments, said sample comprises plasma membranes, cellularmembranes, and/or organellar membranes. In some embodiments, saidinactive enzyme is a cAMP dependent protein kinase. In some embodiments,said detection system comprises a substrate capable of beingphosphorylated by PKA. In some embodiments, said substrate comprises SEQID NO: 1. In some embodiments, said detection system further comprisesan enzyme capable of utilizing ATP to generate a luminescent signalwherein said enzyme is luciferase. In some embodiments, said substratecomprises a radioactively labeled biotinylated substrate furthercomprising SEQ ID NO: 1. In some embodiments, said detection systemfurther comprises a streptavidin coated binding surface. In someembodiments, said substrate comprises a fluorescently labeled substratefurther comprising SEQ ID NO: 1, wherein said fluorescent label ispreferentially rhodamine. In some embodiments, the method of the presentinvention further comprises the addition of one or more inhibitors ofphosphodiesterases, and/or the addition of an agonist or antagonistcapable of affecting adenylyl cyclase activity.

In one embodiment, the present invention provides a method fordetermining phosphodiesterase activity in a sample comprising a samplethat may contain a phosphodiesterase, adding to said sample an inactiveenzyme capable of being activated by cAMP, adding a detection systemcapable of detecting the activity of said activated enzyme andgenerating a detectable signal, and determining phosphodiesteraseactivity present in said sample based on said signal. In someembodiments, said sample comprises a lysate. In some embodiments, thesample lysate is derived from bacteria, yeast or mammalian cells. Insome embodiments, said sample comprises plasma membranes, cellularmembranes, and/or organellar membranes. In some embodiments, saidphosphodiesterase is a cyclic nucleotide phosphodiesterase. In someembodiments, said cyclic nucleotide is cAMP or cGMP. In someembodiments, said inactive enzyme is a cAMP dependent protein kinase ora cGMP dependent protein kinase. In some embodiments, said detectionsystem comprises a substrate capable of being phosphorylated by PKA orPKG. In some embodiments, said substrate comprises SEQ ID NO: 1. In someembodiments, said detection system further comprises an enzyme capableof utilizing ATP to generate a luminescent signal wherein said enzyme isluciferase. In some embodiments, said substrate comprises aradioactively labeled biotinylated substrate further comprising SEQ IDNO: 1. In some embodiments, said detection system further comprises astreptavidin coated binding surface. In some embodiments, said substratecomprises a fluorescently labeled substrate further comprising SEQ IDNO: 1, wherein said fluorescent label is preferentially rhodamine. Insome embodiments, the method of the present invention further comprisesthe addition of one or more inhibitors of phosphodiesterase activity.

In one embodiment, the present invention provides a method fordetermining G-protein coupled receptor activity in a sample comprising asample that may contain a GPCR, adding to said sample an inactive enzymecapable of being activated by cAMP, adding a detection system capable ofdetecting the activity of said activated enzyme and generating adetectable signal, and determining GPCR activity present in said samplebased on said signal. In some embodiments, said sample comprises alysate, more preferably a lysate derived from mammalian cells. In someembodiments, said sample comprises plasma membranes. In someembodiments, said inactive enzyme is a cAMP dependent protein kinase. Insome embodiments, said detection system comprises a substrate capable ofbeing phosphorylated by PKA. In some embodiments, said substratecomprises SEQ ID NO: 1. In some embodiments, said detection systemfurther comprises an enzyme capable of utilizing ATP to generate aluminescent signal wherein said enzyme is luciferase. In someembodiments, said substrate comprises a radioactively labeledbiotinylated substrate further comprising SEQ ID NO: 1. In someembodiments, said detection system further comprises a streptavidincoated binding surface. In some embodiments, said substrate comprises afluorescently labeled substrate further comprising SEQ ID NO: 1, whereinsaid fluorescent label is preferentially rhodamine. In some embodiments,the method of the present invention further comprises the addition ofone or more inhibitors of phosphodiesterase activity and/or addition ofan agonist or antagonist of GPCRs.

In one embodiment, the present invention provides a kit for determiningthe concentration of cyclic nucleotides in a sample comprising a cyclicnucleotide, a protein kinase, ATP, a protein kinase substrate, andinstructions for using said kit in determining said concentration ofsaid protein kinase substrate. In some embodiments, said kit furthercomprises a luminescent detection system. In some embodiments, said kitfurther comprises a fluorescent detection system. In some embodiments,said kit further comprises a radioactive detection system.

In one embodiment, the present invention provides a kit for determiningthe cyclic nucleotide phosphodiesterase activity m a sample comprisingsubstrates from cAMP and cGMP, a protein kinase, a protein kinasesubstrate, and instructions for using said kit in determining saidactivity of said cyclic nucleotide phosphodiesterase. In someembodiments, said kit further comprises a luminescent detection system.In some embodiments, said kit further comprises a fluorescent detectionsystem. In some embodiments, said kit further comprises a radioactivedetection system.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a G-protein coupled receptor signaling pathway. A G-proteincoupled receptor subunit G_(αs) stimulates adenylyl cyclase and cAMP isgenerated from ATP. Cyclic AMP binds to the regulatory subunit of PKAreleasing the catalytic subunits that phosphorylate substrates of PKA.Conversely, an inhibitory GPCR subunit G_(αi) inhibits adenylyl cyclasethereby blocking phosphorylation of PKA substrates. Phosphodiesterasesaffect PKA substrate phosphorylation by hydrolyzing cAMP to AMP and cGMPto GMP, which does not bind to the PKA regulatory subunits.

FIG. 2 is a graph showing that as cAMP concentration increases there isa corresponding decrease in sample luminescence in assays comprisingPKA.

FIG. 3 is a graph showing that as the concentration of forskolin, adirect stimulant of adenylyl cyclase, increases there is a decrease insample luminescence in assays comprising PKA.

FIG. 4A shows a graph demonstrating that as agonists induce the dopaminereceptor D1 (G_(αs)-protein coupled receptor) expressed in D293 cellsthere is a decrease in luminescence in assays comprising PKA and FIG. 4Bshows a graph demonstrating that the addition of an antagonist, in thepresence of an agonist, to dopamine receptor D expressing D293 cellscauses an increase in luminescence in assays comprising PKA.

FIG. 5 shows that as phosphodiesterase II concentration increases in thepresence of cAMP there is an increase in luminescence m assayscomprising PKA, Holoenzyme-R-II α protein kinase A.

FIG. 6 demonstrates that as cyclic nucleotide concentration increases,luminescence increases in assays comprising PKA. Holoenzyme-R-II αprotein kinase A, regardless of whether the cyclic nucleotide is cAMP orcGMP but with different affinities.

FIG. 7A shows a graph demonstrating that as phosphodiesterase Vconcentration increases in the presence of cGMP there is an increase inluminescence in assays comprising PKA, Holoenzyme-R-II α protein kinaseA and FIG. 7B shows a graph demonstrating that as an inhibitor ofphosphodiesterase V, Zaprinast, increases in the presence of cGMP thereis a decrease in luminescence m assays comprising PKA, Holoenzyme-R-II αprotein kinase A.

FIG. 8A and FIG. 8B demonstrate cAMP production in plasma membranepreparations from different mammalian cells prepared using hypotonic ornitrogen cavitation lysis methods; FIG. 8A shows human embryonic kidney(HEK) 293 cells and FIG. 8B shows Chinese hamster ovary (CHO) cells.

FIG. 9 shows the EC₅₀ for Forskolin using 1 μg of DRD1-D293 plasmamembrane preparations; assays comprised PKA to detect cAMP.

FIG. 10 demonstrates D1 receptor activation in plasma membranepreparation following activation by addition of dopamine. Activation ofdopamine receptor was evaluated by measuring cAMP production using PKA.

FIG. 11A shows an exemplary titration of Dopanune D2 receptor with theagonist Quinpirole in the presence of 10 μM forskolin using D2 stablytransfected D293 cells and FIG. 11B shows an exemplary titration of theD2 dopamine D2 receptor antagonists Raclopride in the presence of 100 nMQuinpirole and 10 uM forskolin using D2 stably transfected D293 cells,assays comprised PKA.

DEFINITIONS

As used herein, the term “sample” is used in its broadest sense. In onesense, it is meant to include a specimen or culture obtained from anysource, as well as biological and environmental samples. Biologicalsamples may be obtained from animals (including humans) and encompassfluids, solids, tissues, and gases. Biological samples include bloodproducts, cell lysates, and components of cell lysates. Environmentalsamples include environmental material such as surface matter, soil,water, crystals and industrial samples. A sample may or may not containa substance that modulates cyclic nucleotide concentration. Suchexamples are not however to be construed as limiting the sample typesapplicable to the present invention.

As used herein, the term “agonist” refers to any substance that maystimulate the activity of a receptor, enzyme, or other protein.

As used herein, the term “antagonist” refers to any substance that mayinhibit the activity of a receptor, enzyme, or other protein.

As used herein, the term “substrate” refers to any polypeptide that isacted on by an enzyme or other protein.

As used herein, the term “inhibitor” refers to any compound thatinhibits enzyme activity or biochemical reactions.

As used herein, the term “detection” refers to qualitatively orquantitatively determining the presence or absence of a substance withina sample. For example, methods of detection as described herein include,but are not limited to, luminescence, radioactivity and fluorescence.

As used herein, the term “lysate” refers, in its broadest sense, to thecellular debris and fluid that is released from a cell when the cellmembrane is broken apart, or lysed. For example, as described hereinlysates that find utility in the present invention include, but are notlimited to, lysates from prokaryotic cells such as bacteria, and lysatesfrom eukaryotic cells such as yeast, plant and mammalian cell lysates.Cellular debris that is a product of eukaryotic cellular lysis includes,but is not limited to, organelles (e.g., endoplasmic reticulum, nucleus,ribosomes, mitochondria, etc), cellular structural components such asmicrotubules, plasma membranes, organellar membranes, cellularmembranes, and the like.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the methods of the present invention provide formonitoring the modulations of cellular proteins by monitoring thechanges in activity of a protein kinase due to activation by cyclicnucleotides. Cellular levels of cyclic nucleotides reflect the balancebetween the activities of cyclases and cyclic nucleotidephosphodiesterases (FIG. 1). Cyclic AMP binds to the regulatory subunitsof the tetramer PKA. Cyclic GMP binds to the regulatory subunit of cGMPdependent protein kinase, or PKG. The present invention is not limitedto a particular mechanism. Indeed, an understanding of the mechanism isnot necessary to practice the present invention. Nonetheless, it wasfound that not only does cAMP bind to the regulatory subunits of type IIPKA, but cGMP also binds to type II PKA regulatory subunits. Therefore,once cAMP or cGMP binds to the regulatory subunits of PKA, the PKAactive catalytic subunits are capable of phosphorylatingserine/threonine protein kinase substrates by transfer of a phosphatefrom ATP to the substrate phosphorylation site. As such, PKA activityserves as an indicator of the amount of cAMP or cGMP present in asample.

As previously stated, cyclases and phosphodiesterases directly influencethe amount of cyclic nucleotides present in a sample. For example, whenactivators or inhibitors of adenylyl cyclase are present, cAMPconcentration will increase or decrease, respectively, thereby causingan increase or decrease in PKA activity. The same is found foractivators or inhibitors of guanylyl cyclase. Adenylyl cyclase is partof a signaling pathway associated with GPCRs. An agonist or antagonistof a GPCR will affect the activity of adenylyl cyclase, and thus PKAactivity. Conversely, phosphodiesterases hydrolyze cAMP to AMP and cGMPto GMP, so as agonists or antagonists of this enzyme are present in asample, cyclic nucleotide concentration will decrease or increase,respectively, thereby causing a decrease or increase in PKA activity. Assuch, the methods as described herein provide for monitoring modulationsof cAMP, adenylyl cyclase, cGMP, phosphodiesterases, and GPCRs.

To maximize the event that only cyclic nucleotides in a sample are ableto activate the PKA of the method, the PKA of the method should be aspure of a PKA type II holo-enzyme (e.g., PKA regulatory and catalyticsubunits are associated) as possible. Preferably, the PKA type IIholo-enzyme is substantially free from unassociated active catalyticsubunits. The purity of the PKA holo-enzyme should be sufficient topermit monitoring of the modulation of the cyclases and GPCRs whencompared to a control. Similarly, if PKG is used as described herein, itshould be similarly substantially free from unassociated activecatalytic subunits. To maximize the methods as described herein, the PKAholo-enzyme used for said methods should contain <10% (>90% pure),preferably <5% (>95% pure), more preferably <1% (>99% pure), and mostpreferably <0.1% (>99.9% pure) unassociated active catalytic subunits.Assays to test for the percentage of unassociated active catalyticsubunits are those that, for example, compare the activity of a testsample of PKA holo-enzyme with that of a control sample that containsinactivated holo-enzyme.

One embodiment of the present invention provides for determining theconcentration of cyclic nucleotides in a sample. In some embodiments,the present method may be used to determine the amount of cAMP or cGMPin a sample. A sample of the present method comprises, but is notlimited to, cell culture media, a buffered solution, cells, and celllysates. In some embodiments, said sample comprises a lysate. In someembodiments, the sample lysate is derived from bacteria, yeast ormammalian cells. In some embodiments, said sample comprises plasmamembranes, cellular membranes, and/or organellar membranes.

In one embodiment, to determine the concentration of cyclic nucleotidesin a sample, the present invention comprises a protein kinase, substrateand ATP. In some embodiments, the present invention comprises PKA orPKG, such that as a cyclic nucleotide binds to the regulatory subunitsof the kinase the active catalytic subunits are capable of utilizing ATPin phosphorylating a substrate. In some embodiments, the inventioncomprises a serine/threonine protein kinase substrate that demonstratesan increased affinity for PKA or PKG. In some embodiments, the methodcomprises a substrate comprising the polypeptide sequence LRRASLG (SEQID NO: 1).

Detection methods used to determine the cAMP concentration of a sampleusing the present method includes, but is not limited to, the use ofbioluminescence, chemiluminescence, colorimetry, radioactivity, ordifferential output based on different fluorescence technologies. In oneembodiment, kinase activity is measured in the methods described hereinand any suitable kinase assay can be used. For example, known kinaseassays include, but are not limited to, luminescent assays such asKinase-Glo™ Luminescent Kinase Assay (Promega Corporation, Madison Wis.)and PKLight™ HTS Protein Kinase Assay (Cambrex, N.J.), fluorescentassays such as Kinome™ Hunter (DiscoverX, Fremont Calif.) and HitHunter™FP Kinase Assay (DiscoverX, Fremont Calif.) and ProFluor™ PKA Assay(Promega Corporation, Madison Wis.), and radioactivity assays such asSignaTECT® cAMP-Dependent Protein Kinase (PKA) Assay System (PromegaCorporation, Madison Wis.).

It is contemplated that different luminescent detection methods exhibitdifferent patterns of luminescent output with respect to PKA activity.In one embodiment, a luminescent detection method is a method thatdetects kinase activity. In some embodiments, a luminescent detectionmethod as described herein comprises an enzyme, a substrate, and anappropriate buffer. In some embodiments, the present invention detectschanges in cAMP concentration by bioluminescence. In some embodiments,the present invention detects changes in cAMP concentration by utilizinga luciferase. In some embodiments, the present invention detects changesin cAMP concentration by utilizing a coleopteran luciferase. Forexample, as cyclic nucleotides bind to the regulatory subunits of PKAthe catalytic subunits are able to utilize ATP for substratephosphorylation and ATP is depleted. The coleopteran luciferase enzymeutilizes ATP and other co-factors to convert its cognate substrateluciferin into oxyluciferin, a byproduct of the reaction beingluminescence, or light. As ATP decreases in a sample there is lessavailable for luciferase and a decrease in luminescence is seen. Theluminescent ouput (relative light units or RLUs) is used to detect achange in luminescence of a sample relative to that of a control. Otherluminescent detection methods may exhibit differential light output withrespect to PKA activity.

In one embodiment, the present invention provides a detection systemwhereby cyclic nucleotide concentration in a sample is determined byradioactive means. Different radioactive detection methods may exhibitdifferent patterns of radioactive output with respect to PKA activity.In some embodiments, a radioactive detection method is a method todetect kinase activity. In some embodiments, the radioactive detectionmethod as described herein comprises a modified substrate, a suitablebuffer, and a surface capable of capturing the modified substrate. Insome embodiments, the radioactive method comprises the SignaTECT®cAMP-Dependent Protein Kinase (PKA) Assay System (Promega Corporation,Madison, Wis.). In some embodiments, the radioactive detection methodcomprises radioactive ATP. In some embodiments, the radioactivedetection method comprises γ³²P-ATP or γ³³P-ATP.

In one embodiment, the radioactive detection method further comprises asubstrate capable of being phosphorylated by PKA. In some embodiments,the radioactive detection method comprises a substrate capable of beingphosphorylated by PKA in association with a ligand. In some embodiments,the radioactive detection method comprises a biotinylated substratecomprising the polypeptide sequence LRRASLG (SEQ ID NO: 1). In someembodiments, the detection method as described herein further comprisesa surface upon which resides a compound that captures thesubstrate/ligand. For example, the surface is a membrane that is coatedwith streptavidin. As the cyclic nucleotides bind to the regulatorysubunit of PKA, the catalytic subunits utilize the radioactively labeledATP and transfer a radioactive phosphate onto the substrate therebycausing the substrate to be radioactive. The radioactive ligand-coupledsubstrate is captured on a surface upon which resides a compound thatwill capture the ligand (e.g. strepravidin). In some embodiments, thesurface is washed free of excess radioactivity, and radioactivitycaptured on the capture surface is measured. The radioactive output(counts per unit time) is used to detect a change in radioactivity of asample relative to that of a control.

In one embodiment, the present invention provides a detection systemwhereby cyclic nucleotide concentration in a sample is determined byfluorescent means. Different fluorescence detection methods may exhibitdifferent patterns of fluorescence output with respect to PKA activity.In one embodiment, a fluorescence detection method is a method to detectkinase activity. In some embodiments, the fluorescent detection methodof the present method comprises an enzyme, a modified substrate, and asuitable buffer. In some embodiments, the fluorescent method comprisesthe ProFluorm PKA Assay (Promega Corporation, Madison, Wis.). In oneembodiment, the fluorescent detection method of the present inventioncomprises a fluorophore. In some embodiments, the fluorescent detectionmethod comprises the fluorophore rhodamine-110.

In one embodiment, the fluorescent detection method as described hereinfurther comprises a substrate. In some embodiments, the substrate of thefluorescent detection method comprises the polypeptide sequence LRRASLG(SEQ ID NO: 1). In some embodiments, the fluorescent detection methodcomprises an enzyme. In some embodiments, an enzyme of the fluorescentdetection method as described herein is a protease. In some embodiments,the protease of the fluorescent detection method is capable of digestingthe substrate when it is not phosphorylated.

In one embodiment, the fluorescent detection method comprises asubstrate in association with a fluorophore. In some embodiments, thefluorescent detection method comprises two substrates in associationwith a fluorophore such that as the fluorophore is in association withthe substrates there in decreased fluorescence when compared to afluorophore that is free from association with the substrate. Forexample, as the cyclic nucleotides bind to the regulatory subunits ofPKA the catalytic subunits are capable of phosphorylating a cognatesubstrate. The substrates of the fluorescent detection method arecoupled to a fluorophore such that the fluorophore exhibits decreasedfluorescence when bound to the substrates. A protease as describedherein digests the substrate up to the point of phosphorylation.Therefore, if cyclic nucleotide concentration in a sample is increased,more substrate will be phosphorylated and the fluorescence will remainlow. Conversely, if cyclic nucleotide concentration in a sample is low,then less substrate will be phosphorylated, protease digestion of thenon-phosphorylated substrate will be complete thereby releasing thefluorophore and fluorescence will increase. The fluorescence output(relative fluorescent units) is detected and a change in fluorescence ofa sample relative to that of a control is determined.

In one embodiment, the present invention provides for determining cyclicnucleotide concentration in a sample and correlating the cyclicnucleotide concentration with cyclase activity in a sample. In oneembodiment, the cyclic nucleotides to be detected are cAMP or cGMP. Asample of the present method comprises, but is not limited to, cellculture media, a buffered solution, cells, and cell lysates. In someembodiments, said sample comprises a lysate. In some embodiments, thesample lysate is derived from bacteria, yeast or mammalian cells. Insome embodiments, said sample comprises plasma membranes, cellularmembranes, and/or organellar membranes. In some embodiments, the cyclaseof the present invention is chosen from a group consisting of adenylylcyclase and guanylyl cyclase. In one embodiment, the cyclase of thepresent invention is adenylyl cyclase. In some embodiments, the presentinvention is used to find substances that have an affect on adenylylcyclase activity. For example, methods of the present invention are usedto find substances that either stimulate (e.g. increase) or inhibit(e.g. decrease) adenylyl cyclase activity. Examples of substances thatstimulate adenylyl cyclase activity include, but are not limited to,forskolin and forskolin derivatives such as 7-Deacetyl-forskolin,6-Acetyl-7-deacetyl-forskolin and 7-Deacetyl-7-O-hemisuccinyl-forskolin.Examples of substances that inhibit adenylyl cyclase activity include,but are not limited to; cell permeable inhibitors such as9-(Tetrahydrofuryl)-adenine, 2′, 5′-Dideoxyadenosine and9-(Cyclopentyl)-adenine; competitive inhibitors such as substrateanalogs β-L-2′,3′-Dideoxy-adenosine-5′-triphosphate, β-L-Adenosine5′-triphosphate and Adenosine 5′-(β γ-methylene)-triphosphate;non-competitive inhibitors such as 9-(Arabinofuranosyl)-adenine,9-(Xylofuranosyl)-adenine and 2′,5′-Dideoxyadenosine 3′-tetraphosphate;other inhibitors such asCis-N-(2-Phenylcyclopentyl)azacyclotridec-1-en-2-amine,9-(2-Diphosphorylphosphonylmethoxyethyl)adenine and polyadenylyl.

In one embodiment, to determine the affect of substances on adenylylcyclase activity, the methods of the present invention comprise aprotein kinase, substrate and ATP. In some embodiments, the presentmethod comprises a cAMP dependent protein kinase (PKA) such that as acyclic nucleotide binds to the regulatory subunits of the kinase andreleases the active catalytic subunits that are capable of utilizing ATPin phosphorylating a serine/threonine protein kinase substrate. In someembodiments, the present method comprises a serine/threonine proteinkinase substrate that demonstrates an increased affinity for the freecatalytic subunit of PKA. In some embodiments, the present methodcomprises a substrate comprising the polypeptide sequence LRRASLG (SEQID NO: 1). Adenylyl cyclase generates cAMP from ATP, therefore asubstance which affects adenylyl cyclase activity impacts theconcentration of cAMP in a sample. Detection methods have been describedin previous embodiments, and those detection methods are equallyapplicable here. For example, as substances affect the activity ofadenylyl cyclase in a sample, the cAMP concentration will increase ordecrease, which will cause an increase or decrease in substratephosphorylation via PKA. The detection method output as previouslydescribed is used to determine the increase or decrease in cAMPconcentration that is correlated with an increase or decrease inadenylyl cyclase activity of a sample relative to that of a control.Therefore, when an agonist of an adenylyl cyclase is present in a samplethereby stimulating adenylyl cyclase activity, there is an increase incAMP production that is reflected in the output of the detection methodof use. Conversely, if an antagonist of an adenylyl cyclase is presentin a sample thereby inhibiting adenylyl cyclase activity, there is adecrease in cAMP production, which is reflected in the output of thedetection method of use.

In one embodiment, the present invention determines the concentration ofcyclic nucleotides in a sample and correlates the cyclic nucleotideconcentration with phosphodiesterase activity. In some embodiments, thecyclic nucleotides to be detected are cAMP or cGMP. In some embodiments,if cGMP is the cyclic nucleotide used for detection, then the sample hasan overabundance of cGMP relative to cAMP. In some embodiments, whencGMP is the cyclic nucleotide used for detection, phosphodiesterase IV(a cAMP specific phosphodiesterase) is present in the sample. In someembodiments, a sample of the present invention includes, but is notlimited to, cell culture media, a buffered solution, cells, and celllysates. In some embodiments, said sample comprises a lysate. In someembodiments, the sample lysate is derived from bacteria, yeast ormammalian cells. In some embodiments, said sample comprises plasmamembranes, cellular membranes, and/or organellar membranes.

In one embodiment, the phosphodiesterase is a cyclic nucleotidephosphodiesterase. In some embodiments, the cyclic nucleotidephosphodiesterase activity to be determined, when detecting cAMPactivation of PKA, is from a group consisting of phosphodiesterase II,phosphodiesterase III, and phosphodiesterase IV. In some embodiments,the cyclic nucleotide phosphodiesterase activity to be determined, whendetecting cGMP activation of PKA, is from a group consisting ofphosphodiesterase II, phosphodiesterase II, and phosphodiesterase IV andphosphodiesterase V. In some embodiments, methods of the presentinvention are used to find substances that have an affect onphosphodiesterase activity. In some embodiments, the present inventionis used to find substances that either stimulate (e.g. increase) orinhibit (e.g. decrease) phosphodiesterase activity. An examples of asubstance that inhibits phosphodiesterase II activity includes, but isnot limited to, Erythro-9-(2-hydroxy-3-nonyl)adenine. Examples ofsubstances that inhibit phosphodiesterase II activity include, but arenot limited to,1,6-Dihydro-2-methyl-6-oxo-(3,4′-bipyridine)-5-carbonitrile,1,3-Dihydro-4-methyl-5-(4-methylthiobenzoyl)-2H-imidazol-2-one andTrequisin hydrochloride. Examples of substances that inhibitphosphodiesterase IV activity include, but are not limited to,4-[3-(Cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone,4-(3-Butoxy-4-methoxybenzyl)imidazolidin-2-one and1-Ethyl-4-[91-methylethylidene-hydrazino]1H-pyrazolo[3,4-b]pyridine-5-carboxylicacid ethyl ester hydrochloride. Examples of substances that inhibitphosphodiesterase V activity include, but are not limited to,1,4-Dihydro-5-(2-propoxyphenyl)-7H-1,2,3-triazolo(4,5-d-pyrimidin-7-one(Zaprinist), Dipyridamole and1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl-phenylsulfolyl]-4-methylpiperazinecitrate. An example of a substance that is a non-selective inhibitor ofcyclic nucleotide phosphodiesterases is 3-Isobutyl-1-methylxanthine.

In one embodiment, to determine the affect of substances onphosphodiesterase activity, the present invention comprises cyclicnucleotides, a protein kinase, a substrate and ATP. In some embodiments,the present invention comprises PKA such that as a cyclic nucleotide,either cAMP or cGMP, binds to the regulatory subunits of the kinase arereleased and become capable of utilizing ATP in phosphorylating aserine/threonine protein kinase substrate. In some embodiments, thepresent invention comprises a serine/threonine protein kinase substratethat demonstrates an increased affinity for a cAMP dependent proteinkinase. In some embodiments, the present invention comprises a substratecomprising the polypeptide sequence LRRASLG (SEQ ID NO: 1).

Phosphodiesterases hydrolyze cyclic nucleotides, cAMP to AMP and cGMP toGMP. Detection methods have been described in previous embodiments, andthose detection methods are equally applicable here. For example, assubstances modulate the activity of a phosphodiesterase in a sample, thecyclic nucleotide concentration increases or decreases, thereby causingan increase or decrease in substrate phosphorylation via PKA. Adetection method output as described herein is used to determine theincrease or decrease in cyclic nucleotide concentration that iscorrelated with an decrease or increase in phosphodiesterase activity ofa sample relative to that of a control. Therefore, when an agonist of aphosphodiesterase is present in a sample thereby stimulatingphosphodiesterase activity, there is an increase in hydrolysis of cAMPto AMP or cGMP to GMP, which is reflected in the output of the detectionmethod of use. Conversely, if an antagonist of a phosphodiesterase ispresent in a sample thereby inhibiting phosphodiesterase activity, thereis a decrease in hydrolysis of cAMP to AMP or cGMP to GMP, which isreflected in the output of the detection method of use.

In one embodiment, a method as described herein determines theconcentration of cyclic nucleotides in a sample and correlates thecyclic nucleotide concentration with G-protein coupled receptor (GPCR)activity. In some embodiments, the cyclic nucleotides to be detected arecAMP or cGMP. In some embodiments, a sample of the present inventioncomprises, but is not limited to, cell culture media, a bufferedsolution, cells, and cell lysates. In some embodiments, said samplecomprises a lysate. In some embodiments, the sample lysate is derivedfrom bacteria, yeast or mammalian cells. In some embodiments, saidsample comprises plasma membranes, cellular membranes, and/or organellarmembranes. In some embodiments, methods of the present invention areused to find substances that have an affect on GPCR activity. In someembodiments, the present invention is used to find substances thateither stimulate (e.g. increase) or inhibit (e.g. decrease) GPCRactivity. A representative list of G-protein coupled receptors can befound in Hermans, E., 2003, Pharmacology & Therapeutics 99:25-44,incorporated herein by reference in its entirety. Examples of GPCRinclude, but are not limited to, the dopamine receptor D1 (SEQ ID NO: 2)(U.S. Pat. No. 5,389,543) and the β-2-adrenergic receptor and theprostaglandin E1 receptor. Examples of substances that increase dopaminereceptor D1 (SEQ ID NO: 2) activity include, but are not limited to,dopamine, apomorphine,1-Phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol (SKF 38393) and6-Chloro-7.8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepinehydrobromide (SKF 82958). Other dopamine receptors include, but are notlimited to, D2, D3, D4, and D5 receptors. An example of a substance thatincreases the activity of the β-2-adrenergic receptor includes, but isnot limited to, isoproterenol. An example of a substance that increasesactivity of the prostaglandin E2 receptor includes, but is not limited,CP-533,536. Other prostaglandin receptors includes, but are not limitedto, EP1, EP3 and EP4.

In one embodiment, to determine the affect of substances on GPCRactivity, methods of the present invention comprise cyclic nucleotides,a protein kinase, a substrate and ATP. In some embodiments, the presentinvention comprises PKA such that as a cyclic nucleotide binds to theregulatory subunits of the kinase and active catalytic subunits arecapable of utilizing ATP in phosphorylating a serine/threonine proteinkinase substrate. In some embodiments, the present invention comprises aPKA which upon binding of cyclic nucleotides to its subunits, the kinaseactivity of the catalytic subunit is generated and utilizes ATP forphosphorylating a serine/threonine protein kinase substrate. In someembodiments, the present invention comprises PKA, which upon binding ofcyclic nucleotides to its regulatory subunit, the kinase activity of thecatalytic subunits is generated and utilizes ATP for phosphorylating aserine/threonine protein kinase substrate. In some embodiments, thesubstrate comprises the polypeptide sequence LRRASLG (SEQ ID NO: 1).

G-protein coupled receptors are integral membrane proteins which areinvolved in signaling from outside to inside a cell. There are manydiseases that are caused by GPCR malfunction, therefore the ability ofmethods of the present invention to define whether substances have anaffect on GPCR activity is of importance both academically andclinically. As substances either stimulate or inhibit a G-proteincoupled receptor, the associated adenylyl cyclase is affected whereinits activity will increase or decrease, respectively. As the adenylylcyclase activity is modulated by stimulation or inhibition through theGPCR, the amount of ATP that is converted to cAMP is affected, therebycontrolling the amount of cAMP that is available to associate with theregulatory subunits of PKA, which in turn controls the amount ofsubstrate phosphorylation that occurs in a sample.

Detection methods have been described in previous embodiments, and thosedetection methods are equally applicable here. As substances affect theactivity of a GPCR in a sample, the cyclic nucleotide concentrationchanges accordingly, which causes an increase or decrease in substratephosphorylation via PKA. The detection method output is used todetermine the increase or decrease in cAMP concentration that iscorrelated to an increase or decrease in GPCR activity of a samplerelative to that of a control. Therefore, when an agonist of a GPCR thatis coupled to Gα_(s) is present in a sample, adenylyl cyclase activityis stimutated causing an increase in cAMP generation, which is reflectedin the output of the detection method used.

Conversely, if an antagonist of a GPCR that is coupled to Gα_(s) ispresent in a sample, adenylyl cyclase activity is inhibited and there isa decrease in cAMP generation that is reflected in the output of thedetection method used. When an agonist of GPCR coupled to Gα_(i) ispresent in a sample, adenylyl cyclase activity is inhibited causing adecrease in cAMP generation that is reflected in the output of thedetection method used. When an antagonist of GPCR coupled to Gα_(i) ispresent in a sample, adenylyl cyclase activity is not inhibited,therefore a potential increase in cAMP generation is realized and isreflected in the output of the detection method used.

In one embodiment, the present invention provides a kit comprising oneor more reagents for conducting any method as described herein. In someembodiments, said reagents are sufficient for conducting the methods asdescribed herein. In some embodiments, said kit reagents include, butare not limited to, controls, instructions, buffers, software for dataanalysis, equipment for practicing the detection methods as describedherein, one or more containers comprising one or more reagents forpracticing the methods as described herein, and tissue culture cells. Insome embodiments, said kits comprise cyclic nucleotides, such as cAMP orcGMP. In some embodiments, said kits comprise enzymes such as PKA andPKG. In some embodiments, said kits comprise cell lysis buffers orsolutions. In some embodiments, said kits comprise reaction buffers. Insome embodiments, said kits comprise protein kinase substrates, eitherlyophilized or in solution. In some embodiments, said kits containbuffers and reagents amenable to a particular detection system ormethod, for example, luminescence, fluorescence, or radioactivedetection systems.

The terminology employed herein is for the purpose of description andshould not be regarded as limiting. Further, the embodiments asdescribed herein are exemplary of what is practiced by using the presentkits, methods and compositions. They are not intended to be limiting,and any person skilled in the art would appreciate the equivalentsembodied therein.

EXAMPLES

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

Example 1-Culture of Mammalian Cells

Mammalian cells HEK D293 (human embryonic kidney) were cultured in thefollowing manner for all cell culture related experiments, unlessotherwise stated. Cells were seeded at a density of 5-10,000 cells/wellin a poly-D-lysine coated 96-well white, clear bottom tissue cultureplate (BD BioCoat™ Poly-D-Lysine Multiwell™ Plates). Cell culture mediaconsisted of Dulbecco's Modified Eagles Medium (DMEM) supplemented with10% Fetal Bovine Serum (FBS), 1 U penicillin and 1 mg/ml streptomycin.For the stably expressing dopamine receptor D1 cell line, 500 μg/ml ofneomycin was added to the culture media for selection and maintenancepurposes. Cells were grown at 37° C./5% CO₂ for approximately 24 hoursuntil they were 60-75% confluent at which point transfection, induction,or other cellular manipulations were performed.

Example 2-Determination of cAMP Concentration

This experiment was conducted to demonstrate that the present inventioncan be used to determine cAMP concentration in a sample, and that thepresent invention can be used with a variety of detection technologiesin determining the cAMP concentration of a sample.

Reactions were performed in a poly-D-lysine coated, white, clear bottom96 well plate however reactions can also be performed in a 384 wellplate by decreasing the amount of added reagents proportionately. Higherdensity plates, such as 1536 well plates, can also potentially be usedby scaling down volume additions accordingly.

A three-fold serial dilution of cAMP starting with 25 μM in 2× InductionBuffer (240 mM NaCl, 7.0 mM KCl, 3.0 mM CaCl₂, 2.4 mM MgSO₄, 2.4 mMNaH₂PO₄, 50 mM NaHCO₃, 20 mM glucose, 200 μM 3-Isobutyl-1-methylxanthine(IBMX), 100 μM 4-(3-Butoxy-4-methoxybenzyl)imidazolidin-2-one (RO201724) was made and 10 μl of each dilution was transferred to separatewells of a 96 well plate. To each cAMP dilution well 10 μl of 2×Induction Buffer and 60 μl of PKA/Substrate Reagent (100 ng/wellHoloenzyme-R-II α protein kinase A (BIAFFIN GmbH & Co., Kassel,Germany), 25 μM Kemptide, 1 μM rATP, 20 mM MgCl₂) were added. The sampleplate was incubated at room temperature for 20 minutes followed byaddition of 80 μl of Kinase-Glo™ Reagent (Promega Corporation, MadisonWis.). Luminescence was read 10 minutes after addition of theKinase-Glo™ Reagent and output was recorded as relative light units(RLU, n=2) and plotted against cAMP concentration using GraphPad Prism®Software Version 4.0 (GraphPad Software, San Diego, Calif.).

As can be seen in FIG. 2, as cAMP concentration increases, luminescencedecreases. The same reciprocal response was seen when using both theSignaTECT® PKA Assay System (radioactivity counts, Promega Corporation,Madison Wis.) and the ProFluor™ PKA Assay System (relative fluorescence,Promega Corporation, Madison Wis.). Therefore, cAMP concentration can beestimated in an unknown sample using the present invention and thestandard curve. Similarly, using this standard curve the concentrationof cAMP in cellular extracts of cells that were treated with agonist orantagonist can be estimated.

Example 3-Monitoring Adenylyl Cyclase Activation in the Presence ofForskolin

Reactions were performed in a poly-D-lysine coated, white, clear bottom96 well plate however reactions can also be performed in a 384 wellplate by decreasing the amount of added reagents proportionately. Higherdensity plates, such as 1536 well plates, can also potentially be usedby scaling down volume additions accordingly.

A two-fold serial dilution of 250 μM forskolin in 2× Induction Bufferwas made. D293 cells were grown to confluency as described in Example 1.Media was removed from the cultured cells, they were washed three timeswith Phosphate Buffered Saline (PBS) and 10 μl of each forskolindilution was added to wells of the 96 well D293 cell culture plate. Acontrol was included by adding 10 μl of 2× Induction Buffer withoutforskolin to several wells of D293 cells. Cells with and withoutforskolin were incubated for 15 min. at room temperature, followed bythe addition of 10 μl of 2× Lysis Buffer (80 mM Tris-HCl, pH 7.5, 2 mMEDTA, pH 8.0, 2 mM EGTA, pH 7.2, 0.4% Tergitol® NP-9, 20% glycerol, 100mM NaF, 200 uM Na₃VO₄, 400 uM leupeptin, 40 ug/ml aprotinin, 400 uM1-Chloro-3-tosylamido-7-amino-2-heptanone (TLCK), 400 uM1-Chloro-3-tosylamido-4-phenyl-2-butanone (TPCK), 200 uM4-(2-Aminoethyl)benzenesulfonyl fluoride-HCl (ABSF), 200 uM IBMX, and 4uM rATP (add TPCK last, vortex the buffer prior to adding TPCK to avoidprecipitation and keep lysis buffer on ice). Cells were allowed to lysefor 15-30 min. at 4° C., and complete lysis was verified by microscopicevaluation. After lysis, 601&l of PKA/Substrate Reagent was added toeach well and the reactions were incubated for an additional 20 min. atroom temperature, followed by the addition of 80 μl of Kinase-Glo™Reagent. Luminescence was read 10 minutes after addition of theKinase-Glo™ Reagent and output was recorded as relative light units(RLU, n=2) and plotted against forskolin concentration using GraphPadPrism® Software Version 4.0.

As shown in FIG. 3, as forskolin concentration increases luminescencedecreases, thereby demonstrating that the present invention can by usedto detect an increase in adenylyl cyclase activity. As forskolindirectly stimulates adenylyl cyclase generating cAMP from ATP, cAMP inturn binds to the regulatory subunits of PKA thereby releasing theactive PKA catalytic subunits, which in turn uses ATP to phosphorylatethe Kemptide substrate. As phosphorylation of Kemptide increases, thereis less ATP available to be used by the luciferase enzyme in theKinase-Glo™ Reagent causing a decrease in luminescence. This effect offorskolin on adenylyl cyclase is seen in FIG. 3 as forskolinconcentration increases so does adenylyl cyclase activity that iscorrelated with a decrease in luminescence. Therefore, the presentinvention is capable of utilizing cAMP to monitor the induction ofadenylyl cyclase by a stimulant.

Example 4-Monitoring Dopamine Receptor D1 Activity in Response toAgonists and Antagonists

Experiments were conducted to demonstrate the ability of the presentinvention to determine the effect of agonists and antagonists on GPCRdopamine receptor D1 (DRD1), a Gα_(s) coupled receptor, in mammaliancells.

A D293 cell line stably expressing DRD1 was created, using standardmolecular biological techniques. Briefly, the gene encoding for DRD1(Genbank NM000794) was amplified using polymerase chain reaction from acDNA containing vector (ATCC, HGR213-1) and cloned into the pTarget™mammalian expression vector following the manufacturer's protocol(Promega Corporation, TM044). The cells were grown as in Example 1 andtransfected with pTarget-DRD1 vector 24 hours after seeding. One daypost-transfection, the cells were trypsinized and re-plated at variousdilutions and fresh media was applied containing 500 μg/ml of theselection drug neomycin. Media was changed every 2-3 days until it wasapparent that drug resistant clones were created. Several neomycinresistant clones were selected for further characterization and testedfor a dopamine receptor D1 response. The clones that showed the highestresponse were expanded and frozen stocks created. One of the cloned celllines was used for subsequent testing.

Three-fold dilutions of 10M stock concentrations of the agonistsdopamine, SKF 38393, apomorphine, and SKF 82958 were diluted in 2×Induction Buffer. For testing the antagonist SCH 23390, a two-foldserial dilution of the antagonist SCH 23390 (5 uM) was also made in 2×Induction Buffer containing 100 nM of the agonist SKF 38393.

Reactions were performed in a poly-D-lysine coated, white, clear bottom96 well plate however reactions can also be performed in a 384 wellplate by decreasing the amount of added reagents proportionately. Higherdensity plates, such as 1536 well plates, can also potentially be usedby scaling down volume additions accordingly.

D293 stable cells expressing DRD1 were seeded as described in Example 1.The following day cells were washed 3 times with PBS and 10 μl of eachagonist dilution was added to specific wells. For control reactions, 10μl of 2× Induction Buffer without agonist was used. Induction wasallowed to proceed for 30 min. at room temperature, at which point 10 μlof 2× Lysis Buffer was added to each well. Lysis took approximately20-30 minutes, and complete lysis was verified by microscopicevaluation. Once cells were completely lysed, 60 μl of PKA/SubstrateReagent was added and the reactions were incubated for an additional 20min. at room temperature. After incubation, 80 μl of Kinase-Glo™ Reagentwas added and luminescence read after a 10 min. incubation at roomtemperature. Output was recorded as RLUs (n=2) and was plotted againstagonist concentration using GraphPad Prism® Software Version 4.0. FIG.4A demonstrates that as agonist concentration increases luminescentsignal decreases, thereby showing the inducing effect of each agonist onadenylyl cyclase of DRD1 containing cells. Each agonist has a differenteffect on DRD1 activity as demonstrated in FIG. 4A by the different EC₅₀values for the various agonists.

For testing the inhibition by an antagonist, D293 stable cellsexpressing DRD1 were incubated with 10 μl of the antagonist SCH 23390dilutions in the presence of 100 nM of the agonist SKF 38393, incubatedfor 30 min. at room temperature. Addition of PKA/Substrate Reagent andKinase-Glo™ Reagent and subsequent incubations and readings were carriedout as described above.

Output was recorded as RLUs (n=2) and was plotted against agonistconcentration using GraphPad Prism® Software Version 4.0. FIG. 4B showsthat as antagonist concentration increases luminescence increases aswell. This increase is due to inhibition of adenylyl cyclase by theantagonistic affect of SCH 23390 to the agonist SKF 38393. Therefore,the present invention finds utility in monitoring the effects ofagonists and antagonists on the adenylyl cyclase component of a GPCRcoupled to Gα_(s) pathway.

Example 5-Correlation of cAMP and cGMP with PDE Activity

Experiments were conducted to demonstrate the ability of PKA to monitorchanges in cAMP and cGMP concentrations in the presence of cyclicnucleotides and the cognate cyclic nucleotide phosphodiesterases.Experiments were also conducted to monitor the changes of PDE activityin the presence of activators or inhibitors of cyclic nucleotidephosphodiesterases.

Reactions were performed in white 96 well plates however reactions canalso be performed in a 384 well plate by decreasing the amount of addedreagents proportionately. Higher density plates, such as 1536 wellplates, can also potentially be used by scaling down volume additionsaccordingly.

A serial dilution of Bovine Brain Phosphodiesterase II (Sigma, PDE II),which hydrolyzes cAMP to AMP, was created by diluting a 1 mU stock by1/10 increments (1 mU, 0.9 mU, 0.8 mU, etc.) in 2× Induction Bufferminus IBMX and RO 201724. A 12.51l aliquot of each dilution was added to12.5 μl of a solution containing 50 mM Tris HCl, pH 7.5, 10 mM MgCl₂, 50μM CaCl2, 0.1 mg/ml BSA, 20 μM calmodulin (CaM), and 0.5 or 1.0 μM cAMP,total volume of 25 μl in a 96 well white plate. The enzyme reactionswere incubated at room temperature for 15 min., and the reactionterminated by addition of 12.5 μl of Stop Buffer (40 mM Tris HCl pH 7.5,20 mM MgCl₂, 0.1 mg/ml BSA, 375 μM IBMX and 44M ATP). Following additionof the IBMX solution, 25 μl of PKA/Substrate Reagent was added,reactions were incubated for an additional 20 min, and 50 μl ofKinase-Glo™ Reagent was added. Luminescence was measured 10 min. afteraddition of the Kinase-Glo™ Reagent and output was recorded as RLUs(n=2) and plotted against PDE concentration using GraphPad Prism®Software Version 4.0. As seen in FIG. 5, luminescence increased withincreasing PDE II concentration demonstrating the effect of PDE II oncAMP concentrations. As cAMP is hydrolyzed to AMP by PDE II, cAMPconcentration decreases, thereby causing an increase in luminescence.

To demonstrate the ability of cGMP to activate PKA, side by sidetitrations of cAMP and cGMP were performed using the present assaysystem. Two-fold serial dilutions of cAMP and cGMP (initialconcentration of both 40 μM) were made in Stop Buffer without ATP andIBMX, but supplemented with 2 μM ATP. Twenty-five μl of the dilutionswere aliquoted into white 96-well plates, followed by the addition of 25μl of a PKA/substrate reagent containing 100 ng/well Holoenzyme-R-II αprotein kinase A, 20 μM Kemptide, 40 mM Tris HCl pH 7.5, 20 mM MgCl₂,and 0.1 mg/ml BSA. The reactions were allowed to incubate for 20 min. atroom temperature, and 50 μl of Kinase-Glo™ Reagent was added followed byan additional 10 min. incubation. Luminescence was measured 10 min.after addition of the Kinase-Glo™ Reagent and output was recorded asRLUs (n=2) and plotted against cyclic nucleotide (cNMP) concentrationusing GraphPad Prism® Software Version 4.0. As shown in FIG. 6, as theconcentration of the cyclic nucleotides increases the relative lightunits decrease. FIG. 6 demonstrates the ability of cGMP to bind to theregulatory subunits of PKA thereby releasing the active catalyticsubunits, albeit at a lower affinity than that of cAMP.

Example 6-Monitoring cGMP-PDE (PDE V) Activity

A serial dilution of phosphodiesterase PDE V starting with 50 Uconcentration was created in a solution containing 50 mM Tris HCl, pH7.5, 10 mM MgCl₂, 0.5 mM EGTA, 0.1 mg/ml BSA, and 5 μM cGMP in a totalvolume of 25 μl. The enzyme reaction was allowed to progress for 60 min.at room temperature, followed by the addition of 12.5 μl of Stop Buffer.Twenty-five μl of a PKA/substrate reagent containing 100 ng/wellHoloenzyme-R-II α protein kinase A, 40 μM Kemptide, 40 mM Tris HCl pH7.5, and 30 mM MgCl₂ was added to the reaction wells followed by a 20min. incubation at room temperature. An equal volume (50 μl) ofKinase-Glo™ Reagent was added, the reactions were incubated anadditional 10 min., luminescence was measured and output was recorded asRLUs (n=2) and plotted against phosphodiesterase PDE V concentrationusing GraphPad Prism® Software Version 4.0. As can be seen in FIG. 7A,as phosphodiesterase PDE V concentration increases so does the relativeluminescence of the sample. FIG. 7A demonstrates the ability of thepresent assay to monitor not only phosphodiesterases specific to cAMPhydrolysis, but also those specific to hydrolysis of cGMP.

Example 7—Monitoring the Activity of cGMP-PDE (PDE V) in the Presence ofInhibitors

A titration of the phosphodiesterase PDE V selective inhibitor Zaprinast(Sigma) was performed. A two-fold serial dilution of Zaprinast (20 μM)was made in Stop Buffer without IBMX and ATP, supplemented with 10 μMcGMP. An enzyme solution containing PDE V was also made so that every12.5 μl of the enzyme solution contained 15 U (Stop Buffer without IBMXand ATP) of the enzyme. An aliquot (12.5 μl) of each dilution was addedto the reaction wells and an equal amount of the phosphodiesterase PDEVenzyme solution was added to start the reaction. The plate was incubatedfor 30 min. at room temperature, followed by addition of 12.5 μl of StopBuffer supplemented with 1.5 mM IBMX and 4 μM ATP to stop the reaction.An equal volume of a PKA/substrate reagent containing 100 ng/wellHoloenzyme-R-II α protein kinase A, 40 μM Kemptide, 40 mM Tris HCl pH7.5, 20 mM MgCl₂, and 0.1 mg/ml BSA was added to each well, the platewas incubated for an additional 20 min., and 50 μl of Kinase-Glo™Reagent was added. Luminescence was measured 10 min. after addition ofthe Kinase-Glo™ Reagent and output was recorded as RLUs (n=2) andplotted against Zaprinast concentration using GraphPad Prism® SoftwareVersion 4.0. As shown in FIG. 7B, as the amount of phosphodiesterase PDEV inhibitor Zaprinast increases relative light units decrease. FIG. 7Bdemonstrates the correlation between luminescence and Zaprinastconcentration, thereby demonstrating the utility of the assay indetermining potential inhibitors of the cGMP specific phosphodiesterasePDE V. FIG. 7B further displays the IC₅₀ for Zaprinast calculated inpresent experiment compared to that found in the literature (Turko1998), again demonstrating the utility of the assay to monitor changesin activity of a cognate cGMP phosphodiesterase. Therefore, the presentinvention finds utility in measuring cAMP concentration and the activityof its cognate phosphodiesterase in the presence and absence ofinhibitors, as well as monitoring cGMP concentration and the activity ofits cognate phosphodiesterase in the presence or absence of inhibitors.

Example 8-Detection of cGMP Concentration

To determine cGMP concentration in a biological sample, the sampleinitially should be heated to 95° C. for 5 min, followed by addition ofa cAMP selective phosphodiesterase such as PDE IV and an additionalincubation at room temperature for 30 min. An aliquot of the PKASubstrate Reagent could then be added to the samples and incubated atroom temperature for 20 min., followed by addition of Kinase-Glo™Reagent (Promega Corporation, Madison Wis.). Ten minutes after additionof the Kinase-Glo™ Reagent, luminescence would be measured and outputrecorded (RLU) and plotted against different sample volumes. The amountof cGMP in a sample could then be determined using a graphing programsuch as GraphPad Prism® Software Version 4.0. and comparing sampleluminescent output with that of a cGMP standard curve.

To measure activity of a cAMP or cGMP phosphodiesterases in a biologicalsample, the sample should be dialyzed to remove endogenous cAMP andcGMP, for example with a dialysis membrane with a 500 Da cut off. Thesample that remains in the dialysis membrane would be used in thesubsequent experiments. The sample would be incubated with substratesand reagents as described in the previous examples. Thus, for cAMP orcGMP phosphodiesterases, the substrates cAMP or cGMP, respectively,would be used. Detection methods could be used as previously described,and activity of the phosphodiesterase determined by comparing thedetection output with that of a control.

Example 9-Detection of cAMP in Plasma Membranes

This experiment provides an exemplary method for preparing plasmamembrane preparations using hypotonic or nitrogen cavitation lysismethods.

For hypotonic lysis, 3×10⁷ cells were collected by centrifugation at500×g for 5 minutes and washed twice in PBS. The cell pellet wasresuspended in 10 ml of hypotonic lysis buffer (1 mM HEPES pH 7.5, 1 mMEDTA, 0.2 mM leupeptin and 40 μg/ml aprotinin), and the cell suspensionwas homogenized (20 strokes) using a Pyrex dounce homogenizer. In somecases, the cell suspension was homogenized three times to initiatehypotonic cell lysis. To adjust the hypotonicity in the cellsuspensions, HEPES and glycerol were added to a final concentration of25 mM and 10%, respectively, in some of the cell suspensions. To others,an equal volume of 2× buffer A (2×: 50 mM HEPES pH 7.5, 2 mM EDTA, 0.5Msucrose, 0.4 mM leupeptin and 80 μg/ml aprotinin) was added. Allsuspensions were homogenized by an additional 17 strokes.

For nitrogen cavitation lysis, 3×10⁸ cells were collected bycentrifugation at 500×g for 5 minutes and washed twice in PBS. The cellpellet was resuspended in 15 ml of buffer A (0.25 mM sucrose, 25 mMHEPES pH 7.5, 1 mM EDTA, 0.2 mM leupeptin and 40 μg/ml aprotinin). Cellsuspensions were pre-equilibrated in a nitrogen cavitation bomb (ParrInstrument Company, Moline, Ill.) for 20 minutes at 350 Psi, thepressure was slowly released and the cell lysate collected.

For both lysis methods, plasma membrane fractions were collected by thefollowing procedure. The cell lysates were subjected to low speedcentrifugation (1000×g) for 10 minutes to remove cellular debris.Supernatants were collected and subjected to highspeed centrifugation(50,000×g) for 30 minutes to collect the plasma membranes. Plasmamembrane fractions were resuspended in either buffer A, buffer B (25 mMHEPES pH 7.5, 1 mM EDTA, 0.2 mM leupeptin and 40 g/ml aprotinin), orbuffer B containing a final concentration of 10% glycerol.

Different preparation methods were evaluated to determine the optimalmethod whereby membrane integrity is maintained and, most importantly,allowed receptor-G protein-adenylyl cyclase complexes to remain intact.FIG. 8 shows data from the testing of different membrane preparationsusing the different lysis methods and buffers on different cell types.Although the membrane preparations showed induced cAMP production,membranes lysed only by hypotonic lysis and resuspended in buffer Bshowed lower response than membranes lysed in buffer B containingglycerol or buffer A containing sucrose. No difference between themembrane preparations resuspended in buffer B containing glycerol andbuffer A containing sucrose was seen.

Example 10—Detection of Forskolin Stimulated Adenylyl Cyclase Activityin Plasma Membranes

Reactions were performed in a poly-D-lysine coated, white, clear bottom96-well plate, however reactions can also be performed in a 384-wellplate by decreasing the amount of added reagents proportionately. Higherdensity plates, such as 1536-well plates, can also potentially be usedby scaling down volume additions accordingly.

A two-fold serial dilution of 250 μM forskolin in Stimulation Buffer (25mM HEPES pH 7.5, 10 mM MgCl₂, 100 μM IBMX, and 0.1% Tween-20) was made.Plasma membrane preparations were prepared from the HEK293 cells stablyexpressing DRD1 as described in Example 4, according to the method asdescribed in Example 9. Plasma membranes (1 μg of protein in 25 μl inStimulation Buffer) and 10 μM GDP were added to the wells of a 96-wellplate and incubated at room temperature for 10 minutes. Fifteenmicroliters of each forskolin dilution was added to the plasma membranepreparations. A control was included by adding 15l of Stimulation Bufferwithout forskolin to several wells containing pre-incubated plasmamembrane preparations. Plasma membrane preparations with or withoutforskolin were incubated for 15 minutes at room temperature. To detectcAMP production, 40 μl PKA/Substrate Reagent was added to each well andthe reactions were allowed to incubate for an additional 20 minutes atroom temperature, followed by addition of 80 μl of Kinase-Glo™ Reagent.Luminescence was read 10 minutes after addition of the Kinase-Glo™Reagent and output was recorded as relative light units (RLU) andplotted against forskolin concentration using GraphPad Prism® SoftwareVersion 4.0.

As shown in FIG. 9, as forskolin concentration increases, luminescencedecreases, thereby demonstrating that the present invention findsutility in detecting forskolin-stimulated adenylyl cyclase activity inplasma membrane preparations. Therefore, the present invention iscapable of detecting cAMP generation in plasma membrane preparationsupon induction of adenylyl cyclase by a stimulant.

Example 11—Monitoring of Dopamine Receptor D1 Activity in Response toAgonists in Plasma Membranes

Experiments were conducted to demonstrate the ability of the presentinvention to determine the affect of agonists on adenylyl cyclaseactivity on the GPCR dopamine receptor D1 (DRD1) in plasma membranepreparations.

Two-fold dilutions of 10 μM stock concentrations of the agonistsdopamine and SKF38393 were diluted in Stimulation Buffer containing 50μM ATP and 0.2 μM GTP. Two-fold dilutions of a 10 μM stock concentrationof the non-specific ligand for DRD1, quinpirole, were also made.

Reactions were performed in a poly-D-lysine coated, white, clear bottom96-well plate, however reactions can also be performed in a 384-wellplate by decreasing the amount of added reagents proportionately. Higherdensity plates, such as 1536-well plates, can also potentially be usedby scaling down volume additions accordingly.

Plasma membrane preparations were prepared from the HEK293 cells stablyexpressing DRD1 as described in Example 4, according to the method asdescribed in Example 7. Plasma membranes (1 μg of protein in 25 μl ofStimulation Buffer) and 10 μM GDP were added to the wells of a 96-wellplate and incubated at room temperature for 10 minutes. Twentymicroliters of each compound dilution was added to specific wells.Induction was carried out for 15 minutes at room temperature followed byaddition of 40 μl of PKA/Substrate Reagent. The reactions were allowedto incubate for an additional 20 minutes at room temperature followed byaddition of 80 μl of Kinase-Glo™ Reagent. Luminescence was read 10minutes after addition of the Kinase-Glo™ Reagent and output wasrecorded as relative light units (RLU) and plotted against forskolinconcentration using GraphPad Prism® Software Version 4.0.

FIG. 10 demonstrates that as agonist concentration increases luminescentsignal decreases in the case of known specific DRD1 agonist (dopamineand SKF38393) but not in the case of quinpirole, a nonspecific ligandfor DRD1 receptor, thereby showing that activation of DRD1 receptor canbe detected in membrane preparations. Therefore, the present inventionfinds use in monitoring agonist/antagonist induced GPCR receptoractivation in plasma membrane preparations by measuring changes in cAMPconcentration.

In a similar fashion as found in Example 4, experiments with antagonistsof DRD1 can be performed. For example, for testing the inhibition by anantagonist on DRD1 in plasma membranes, 10l of the antagonist SCH 23390dilutions in the presence of 100 nM of the agonist SKF 38393 are addedto DRD1 containing plasma membrane preparations, and the reactions areincubated for 30 min. at room temperature. The addition of PKA/SubstrateReagent and Kinase-Glo™ Reagent and subsequent incubations and readingscan be carried out as described above.

Example 12-Monitoring Dopamine Receptor D2 (DRD2) Activity in Responseto Agonists and Antagonists

Experiments were conducted to demonstrate the ability of the presentinvention to determine the effect of agonists and antagonists on GPCRdopamine receptor D2 (DRD2), a Gα_(i) protein coupled receptor.

A D293 cell line stably expressing DRD2 was created, using standardmolecular biological techniques as described in Example 4 for DRD1.

Cells were briefly washed with phosphate-buffered saline solution toremove traces of serum and were incubated in 20 μl (96 well plate) or7.5 μl (384 well plate) with various concentrations of D2-receptoragonists in the presence of 10 uM Forskolin in Krebs Ringer Buffer (100uM IBMX and 100 uM Ro-20-1724). After 15 minutes of incubation at roomtemperature, cells were lysed using 20 μl (96-well plate) or 7.5 μl(384-well plate) of lysis buffer. After lysis for 15 minutes at RT, akinase reaction was performed using 40 μl reaction buffer containing PKA(40 μl in 96 well and 15l in 384 well plate), and the kinase reactionwas carried out for 20 minutes at room temperature. At the end of thekinase reaction an equal volume of Kinase Glo™ reagent was added andincubated for 10 min at RT, and the plates were read using aluminometer.

For the antagonist based assay, cells were incubated with 10 μMForskolin and EC₅₀ concentration of agonists of D2 receptor in KrebsRinger Buffer that contains 100 μM IBMX and 100 μM Ro-20-1724 and withor without antagonists and the assay was processed as previouslydescribed. As shown in FIG. 11A, an EC₅₀ of 0.5 nM for quinpirole wasobtained which is similar to that reported in the literature. Similarexperimental design was used to test antagonists, except that cells wereincubated with 10 μM forskolin and 100 nM of the D2 agonist qunipirole,and with increasing concentrations of the antagonist raclopride. Asshown in FIG. 11B, an IC₅₀ value of 0.8 nM for raclopride was obtainedwhich is similar to that reported in the literature. The dopamine D2receptor is a Gα_(i) protein coupled receptor. Thus, this assay is notonly capable of monitoring the modulation of Gα_(s) protein coupledreceptors but also the Gα_(i) protein coupled receptors, therebydemonstrating the utility of the assay for HTS screening programssearching for modulators of both classes of receptors.

All publications and patents mentioned in the present application areherein incorporated by reference. Various modification and variation ofthe described methods and compositions of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific preferred embodiments, it should be understoodthat the invention as claimed should not be unduly limited to suchspecific embodiments. Indeed, various modifications of the describedmodes for carrying out the invention that are obvious to those skilledin the relevant fields are intended to be within the scope of thefollowing claims.

1. A method for determining the cyclic nucleotide phosphodiesteraseactivity in a sample, comprising: (a) contacting a sample which maycontain cyclic nucleotide phosphodiesterase activity with (I) aninactive enzyme, wherein said inactive enzyme is Protein Kinase A, (II)ATP, (III) a cyclic nucleotide, and (IV) a detection system capable ofdetecting the activity of activated Protein Kinase A and generating adetectable signal, wherein said detection system comprises a substratecapable of being phosphorylated by Protein Kinase A; and (b) determiningthe phosphodiesterase activity present in said sample based on saidsignal.
 2. The method of claim 1, wherein said sample comprises apurified cyclic nucleotide phosphodiesterase (PDE) enzyme.
 3. The methodof claim 1, wherein said substrate comprises SEQ ID NO:1.
 4. The methodof claim 1, wherein said detection system further comprises an enzymecapable of utilising ATP to generate a luminescent signal.
 5. The methodof claim 4, wherein the enzyme is a luciferase.
 6. The method of claim1, wherein said substrate comprises a fluorescently-labelled substrate.7. The method of claim 1, further comprising the addition of aninhibitor of phosphodiesterase activity.
 8. The method of claim 1,wherein the cyclic nucleotide is cGMP or cAMP.
 9. The method of claim 1,wherein the sample comprises a lysate.
 10. A method for determining theamount of cGMP in a sample comprising: (a) contacting a sample which maycontain cGMP, with an inactive enzyme capable of being activated bycGMP, wherein said inactive enzyme is Protein Kinase A; and ATP; and adetection system capable of detecting the activity of activated ProteinKinase A and generating a detectable signal, wherein said detectionsystem comprises a substrate capable of being phosphorylated by ProteinKinase A; and (b) determining the amount of cGMP present in said samplebased on said signal.
 11. The method of claim 10, further comprisingdetermining adenylyl cyclase activity, phosphodiesterase activity, orG-protein coupled receptor activity in the sample.
 12. The method ofclaim 10, wherein the sample comprises a purified protein kinase A,adenylyl cyclase, phosphodiesterase (PDE), or G-protein coupled receptorenzyme.
 13. The method of claim 10, wherein the sample comprises alysate.
 14. The method of claim 10, wherein said substrate comprises SEQID NO:
 1. 15. The method of claim 10, wherein said detection systemfurther comprises an enzyme capable of utilising ATP to generate aluminescent signal.
 16. The method of claim 15, wherein the enzyme is aluciferase.
 17. The method of claim 10, wherein said substrate comprisesa fluorescently-labelled substrate.
 18. The method of claim 10, furthercomprising the addition of one or more inhibitors of phosphodiesteraseactivity.
 19. The method of claim 10, further comprising the addition ofan agonist or antagonist substance capable of affecting cyclicnucleotide amounts in said sample.
 20. The method of claim 19, whereinsaid agonist or antagonist modulates adenylyl cyclase activity,G-protein coupled receptor activity, or phosphodiesterase activity.